bioRxiv preprint first posted online Jul. 19, 2019; doi: http://dx.doi.org/10.1101/709014. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. H3K4me3 is neither instructive for, nor informed by, transcription. Struan C Murray1, Philipp Lorenz1, Françoise S Howe1, Meredith Wouters1, Thomas Brown1, Shidong Xi1, Harry Fischl1, Walaa Khushaim1, Joseph Regish Rayappu1, Andrew Angel1 & Jane Mellor1* 1Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK. *Corresponding author. Tel: +44 1865 613241; E-mail: [email protected] Key words histone modification; gene regulation; H3K4me3; chromatin; RNA polymerase Abstract H3K4me3 is a near-universal histone modification found predominantly at the 5’ region of genes, with a well-documented association with gene activity. H3K4me3 has been ascribed roles as both an instructor of gene expression and also a downstream consequence of expression, yet neither has been convincingly proven on a genome-wide scale. Here we test these relationships using a combination of bioinformatics, modelling and experimental data from budding yeast in which the levels of H3K4me3 have been massively ablated. We find that loss of H3K4me3 has no effect on the levels of nascent transcription or transcript in the population. Moreover, we observe no change in the rates of transcription initiation, elongation, mRNA export or turnover, or in protein levels, or cell-to-cell variation of mRNA. Loss of H3K4me3 also has no effect on the large changes in gene expression patterns that follow galactose induction. Conversely, loss of RNA polymerase from the nucleus has no effect on the pattern of H3K4me3 deposition and little effect on its levels, despite much larger changes to other chromatin features. Furthermore, large genome-wide changes in transcription, both in response to environmental stress and during metabolic cycling, are not accompanied by corresponding changes in H3K4me3. Thus, despite the correlation between H3K4me3 and gene activity, neither appear to be necessary to maintain levels of the other, nor to influence their changes in response to environmental stimuli. When we compare gene classes with very different levels of H3K4me3 but highly similar transcription levels we find that H3K4me3-marked genes are those whose expression is unresponsive to environmental changes, and that their histones are less acetylated and dynamically turned-over. Constitutive genes are generally well-expressed, which may alone explain the correlation between H3K4me3 and gene expression, while the biological role of H3K4me3 may have more to do with this distinction in gene class. Introduction Histones are universally recognised as an integral component of eukaryotic gene regulation. Histone octamers bind DNA to form nucleosomes, winding the DNA and changing its binding surface, fundamentally altering its accessibility and structure. The extruding tails of the histone proteins can also undergo extensive covalent modification. These histone modifications can alter nucleosome structure and recruit proteins involved in gene regulation, and so can play a role in the modulation of gene expression. However, the extent bioRxiv preprint first posted online Jul. 19, 2019; doi: http://dx.doi.org/10.1101/709014. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. to which these modifications are intrinsic or essential to physiological gene expression and regulation is not well understood. Arguably the most well-studied histone modification is the trimethylation of lysine 4 of histone H3 (H3K4me3). Inputting the search term ‘H3K4me3’ into Pubmed yields nearly 1800 hits, while ‘H3K27’ yields 1315 hits, ‘H3K36me3’ 380, with other histone residues and modifications yielding far less. One likely reason for H3K4me3’s popularity is its well- established association with gene expression (Santos-Rosa et al., 2002). The levels of H3K4me3 at an averaged or ‘meta’ gene show a pronounced peak immediately downstream of the transcription start site (TSS) that rapidly tails off (Pokholok et al., 2005). The height of this peak is well correlated with the level of gene expression (Howe et al., 2017). As a result of this association, H3K4me3 is often referred to as an ‘activating’ histone modification, with a rise in H3K4me3 expected to cause a subsequent rise in gene expression. However, whether the association of H3K4me3 with gene expression is the result of a causal relationship whereby H3K4me3 instructs transcription has never been firmly established (Howe et al., 2017). Like numerous other histone modifications, H3K4me3 is capable of recruiting proteins to chromatin, which could result in the upregulation of transcription. Notably, H3K4me3 has been shown to interact with the general transcription factor TFIID, meaning it could serve to stabilise the formation of the pre-initiation complex (Lauberth et al., 2013; Vermeulen et al., 2007). H3K4me3 binds to a number of different characteristic protein folds, including the PHD finger, the Tudor domain, and the chromodomain (Ruthenburg et al., 2007). Such folds can be found in a range of chromatin remodelling and histone modifying factors, and so could reasonably be expected to mediate a change in transcription in response to changing levels of H3K4me3. There is also evidence that H3K4me3 can itself result as a consequence of transcription. For example, Vastenhouw et al. (2010) reported that the bulk of H3K4me3 occurs after the start of transcription during zygotic activation in zebra fish. Trimethylation of H3K4me3 has been shown to depend upon the phosphorylation of the C-terminal domain of RNA Polymerase II (RNAPII), specifically at Serine 5 (Lee and Skalnik, 2008; Ng et al., 2003). This phosphorylation is deposited by TFIIH, with a sharp peak present at the 5’ end of the gene body (Ng et al., 2003). This could in turn explain why H3K4me3 is predominantly found at the 5’ end of genes. Indeed, covalently fusing the Set1 protein required for H3K4 methylation to RNAPII results in an extended H3K4me3 peak into the body of highly expressed genes (Soares et al., 2017). What might the purpose of this H3K4me3 deposition be? Ng et al. (2003) proposed that H3K4me3 might act as a sort of transcriptional memory, based on the observation that H3K4me3 levels at the GAL10 gene fall away much more gradually than RNAPII following gene inactivation – the implication being that the hypothesised activatory nature of H3K4me3 might allow for a recently repressed gene to become quickly active again. H3K4 methylation is performed by highly conserved enzymes containing the SET methyltransferase domain. These enzymes are present within complexes with other regulatory proteins - the SET1 complexes in S. cerevisiae and humans. One of these subunits – Spp1 and CFP1 in S. cerevisiae and humans respectively – is necessary for maximal levels of H3K4me3; ablation of these proteins specifically and drastically reduce H3K4me3 with little effect on H3K4me1 and H3K4me2 levels (Morillon et al., 2005; Schneider et al., 2005; Thomson et al., 2010). In S. cerevisiae, deletion of Spp1 reduces global levels of H3K4me3 to ~25% (Howe et al., 2017; Sommermeyer et al., 2013). bioRxiv preprint first posted online Jul. 19, 2019; doi: http://dx.doi.org/10.1101/709014. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. We sought to test the hypothesis that H3K4me3 is universally instructive for gene expression. To achieve this, we took advantage of the above observation that deletion of Spp1 reduces H3K4me3 without affecting related marks in S. cerevisiae. We assessed the effects of lowered H3K4me3 at multiple levels of gene expression by utilising a range of experimental and modelling-based approaches. By determining the rates of transcription initiation, elongation and mRNA degradation, the levels of steady-state mRNA and protein, and other gene parameters like expression noise, we find that H3K4me3 is not instructive for gene expression on a genome-wide scale. Conversely, by depleting RNA Polymerase II from the nucleus, we find that the pattern and total level of H3K4me3 genome-wide remains unchanged after an hour, while changes in H3K4me3 associated with environmental stress are small and unrelated to larger changes in transcription. Taken together, our findings demonstrate that transcription and H3K4me3, though well correlated, do not inform and are not required to maintain the levels of the other. Results H3K4me3 predominantly marks highly expressed, environmentally insensitive genes We wanted to ask whether H3K4me3 modulates gene expression at any level. To this end we deleted the SPP1 gene, in order to abrogate H3K4me3 levels genome-wide. We used ChIP- seq to assess H3K4me3 levels in both wild-type and spp1Δ strains. Importantly, we spiked-in our S. cerevisiae cells with known quantities of S. pombe prior to sequencing, so that our data could be properly normalised, and so allowing for direct comparisons between strains. Our H3K4me3 ChIP-seq data was in good agreement with existing genome-wide datasets, with the same distinctive 5’ peak typical of H3K4me3 meta-gene profiles (Fig 1A) (Pokholok et al., 2005). As expected, levels of H3K4me3 are drastically reduced in spp1Δ (Fig 1A-B). The total genome-wide read-count, after normalising to account for the S. pombe spike-in, was ~25% of what it was in wild-type (Fig 1B). This is a similar reduction to what we observe globally by western blot (Fig 1C) and at the FMP27 gene by RT-PCR (Fig D). This reduction is fairly uniform genome-wide, evidenced by the fact that despite this huge reduction, the levels of H3K4me3 in spp1Δ still correlate well genome-wide with the levels in wild-type (Fig 1E-G, spearman correlation coefficient = 0.68).